This invention relates to cell culture inserts for insertion into multi-well plates for culturing cells. More particularly, this invention relates to such inserts which have placed on the bottom surface thereof a microporous membrane as the substrate for the cells being cultured. Even more particularly, this invention relates to such a cell culture insert having an arrangement for breaking away the bottom supporting portion of the membrane, once the cells have been properly developed, as required, so as to remove the supporting structure of the insert to eliminate its presence during subsequent examination of the cells having been developed in the insert.
That is, conventional instrumentation requires a certain dimension for examination of the cells, and eliminating this support mechanism of the cell insert allows much more ready examination of the cells without interference from the removed support structure.
Initially, when cell inserts were developed for multi-well plates, they were comprised of a plastic material with a membrane on the bottom surface thereof, for propagating cells. The inserts were placed in a conventional multi-well plate in the individual compartments therefor. Certain problems arise, however, with such structures in that the inserts have a tendency to move within the confines of the wells. Multi-well plates of the kind discussed here are similar to those shown and claimed in U.S. Pat. No. 4,349,632, for example, which is hereby incorporated by reference in its entirety.
Thus, structures were developed in the form of overhangs positioned around the upper edge of the cell inserts for suspending the cell inserts in the individual openings of the multi-well plates. Representative of such cell inserts are those described and claimed in European Patent Application No. 0239697 filed Oct. 31, 1986 and U.S. Pat. No. 4,871,674 issued Oct. 3, 1989.
While both of these structures have the effect of supporting the inserts in a suspended fashion in the multi-well plates so that the inserts do not move from one side to the other during propagation of the cells, they do interfere, in the sense of ultimate examination of the developed cells, because the supporting structures interfere with some conventional examination instrumentation. These instruments are expensive and not easily replaced. It is to this problem that this invention is particularly directed.
With this invention, by contrast, the bottom portion of the cell insert is arranged to be broken away. For this reason, the cell membrane itself with the developed cells on the surface thereof is supported in a separated structure of simplified configuration allowing for ready examination of the membrane and the cells developed thereon. There is no interference from the "superstructure" so to speak.
This arrangement is most important because it is necessary to maintain the suspension structure of cell inserts, as discussed above to avoid cultured cell damage. That is, with the use of a suspended microporous membrane, two cell types, for example, can be cultured, one of each side of the membrane in the same well. Without suspension, cells on the bottom surface of the membrane would be exposed to damage. The microporous membrane allows free passage of macromolecules, proteins and ions. As a result, the interactions of the two cell types can be studied without actual physical contact between the two cell populations in the suspended state of the insert. The growth environment mimics the in vivo state of cells being developed in vitro and may replace in vivo testing which has taken place in the past.
Moreover, because cells are being cultured on a microporous membrane substrate, they may be directly examined during the culturing. That is, direct viewing is possible of the living cells with phase contrast microscopy, during the actual cultivation period.
As purely representative of materials which may be utilized for the microporous membranes in the device of the invention include, for example, polycarbonate, polyethylene terephthalate. The porosity of the membrane is developed to allow for selective permeation, and as discussed above, the membrane material and/or the degree of porosity is developed to allow for direct viewing with phase contrast microscopy. The membranes are preferably transparent or translucent.
As a further feature of the advance in recent years of the desire to grow cells on porous culture inserts is so that a researcher has access to both the top and bottom surfaces of cells. Solid surfaces do not allow polar cells to grow and function in a normal in vivo fashion because polar cells, for example, have a distinct top and bottom surface. Use of culture inserts with a suspended porous membrane surface allows for this application. Moreover, and as mentioned above, co-culturing can take place with two or three different types of cells physically separated by the porous membrane and/or culture media. The membrane allows molecules and solution to pass the membrane but prevents direct cell contact between the cell types. Of course, as practitioners-in-the-art are aware, a third cell type may be cultured in the well into which the insert is placed.
It follows, that the support is necessary for obtaining all of the uses of the cell insert. Nevertheless, the support interferes with subsequent detailed examination of the cells. Thus, with the invention here, once The cells have been developed on the surface of the porous membrane, the structure immediately supporting the membrane may be broken away, in accordance with this invention, so that the cells may be readily examined in conventional instrumentation.
Other objects and advantages of this invention will be apparent from the following description, the accompanying drawings and the appended claims.